Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for malignant transformation. The most prominent example of this approach is use of a monoclonal antibody (mAb) trastuzumab antibody-drug conjugate, T-DM1 for treatment of Human Epidermal Growth Factor Receptor 2 (HER2) positive cancers. Elevated HER2 expression is associated with increased proliferation and survival of cancer cells and, thereby, contributes to poor therapy outcomes. Given that the efficacy of the mAb depends on overexpression of its target on tumor cells, development of techniques to assess the receptor expression is extremely important for monitoring the efficacy of therapy, allowing one to optimize the treatment. Current clinical evaluation of HER2 expression is based on Immunohistochemistry (IHC) or Fluorescence in situ hybridization (FISH) staining of biopsied tissue. Both methodologies are ex vivo techniques and, due to tumor heterogeneity, may deliver false-positive or -negative results and can hardly be used to monitor therapy. On the other hand, molecular imaging, using HER2-specific fluorescently-labeled probes, allows assessing the status of HER2 receptors in vivo and following treatment in real time. After injection, such probes concentrate preferentially at the sites of interest (e.g., in the tumor), providing quantitative information on HER2 expression in vivo. Probe accumulation at the tumor cells can occur in parallel to the delivery of some widely used HER2 - specific drugs, such as, Trastuzumab, to the diseased site. As a result, HER2 expression can be monitored without interference with the treatment itself, making this contrast agent suitable for an image and treat paradigm. According to our compartmental kinetic model, the quantitative information about HER2 expression level can be extracted from the time dependence of fluorescence intensity, evaluated from the time series of images. Normalized rate of accumulation (NRA) of HER2 specific ligands in the tumor that takes into account the initial concentrations of the free probe ligands in blood is an indicator of the total concentration of HER2 receptors in the tumor. This assumption is supported by our observations that obtained values of NRA are proportional to overexpression of HER2 of the corresponding tumor type, as measured by ELISA and other methods. We have also showed that ABD-Affibody based probe, asymptotic values of fluorescence intensities after similar normalization are proportional to average HER2 expression for different types of breast carcinomas. However, for probes with much faster kinetics, as those based on monomer Affibody, the relationship between concentration of available HER2 receptors and asymptotic fluorescence intensities is not that straightforward, since a large fraction of receptors can be left unbound for the lack of free fluorescent ligands at longer times , where &#61556; is washout time. We found that for probes with fast washout time, such as HER2-specific Affibody-DyLight conjugates, fluorescence imaging based on repeated injections can provide quantitative assessment on HER2 expression level in HER2-positive carcinoma xenografts. This information is extracted from time series of fluorescent images, analyzed in the framework of kinetic model, developed to describe probe binding after each injection. The results show linear correlations between our reconstructed values for HER2 expression with HER2 expression measured by ELISA ex vivo assays in the same tumors. Additional information about the tumor characteristics can be extracted from the lifetime fluorescence measurements, including binding affinity of the probe to cancer cells and environmental conditions (e.g., pH) at a given site, based on local variations in the lifetimes for specially designed probes. We have continued our studies on in-vivo fluorescence lifetime imaging to monitor the efficacy of treatment. Our preliminary results indicate significant correlation between measured fluorescence lifetimes and the outcome of the treatment in the mouse model of human carcinoma. We started a new project for detection of basal cell carcinoma (BCC), the most common skin cancer in human in collaboration with dermatology department at Memorial Sloan Kettering Cancer Center. In the first phase, the affinity and sensitivity of targeted NIR fluorescence probe was tested in-vitro on cancer cell lines with high and no- expression of BerEP4, which is the most expressed biomarker in BCC. In collaboration with NICHD (Dr. Stratakis), we are recruiting Cushings patients for optical imaging before and after surgery. Preliminary results based on five patients with Cushing diagnostics were presented in Annual Meeting of Endocrinology (ENDO) 2013, San Francisco. Four of the 5 patients were surgically cured of disease with post-operative measurements of plasma cortisol. The early results by multispectral imaging (MSI) on the right cheek of the cured patients prior to surgery and a mean of 7 3 days after surgery showed a significant decrease in total blood volume when measured before and after surgery. In contrast, for the only patient in our sample, who was not surgically cured, the fraction of blood volume increased from pre to post-operative measurements. In this pilot study, preliminary data indicate that a decrease in facial plethora after surgery correlates with cure of Cushings. We are pursuing the Kaposi Sarcoma (KS) studies in ongoing clinical trials under four different NCI protocols and therapeutic agents. The goal is to further evaluate diffuse multispectral imaging as a potential supplement to existing response assessment in KS, providing an early non-invasive marker of treatment efficacy. In our preliminary results, multi-spectral images of Kaposi Sarcoma skin lesions were taken over the course of treatment, and blood volume and oxygenation concentration maps were obtained through Principal Component Analysis (PCA) of the data. Corresponding images were compared with clinical and pathological assessment, provided by conventional means. In agreement with our hypothesis that successful treatment would decrease the blood volume in the lesions, the normalized standard deviation for blood volume decreased in each of the 5 patients whose lesion responded to treatment, while the normalized standard deviation for blood volume increased in the 2 patients, whose lesion did not respond to therapy. These initial results confirm that concentrations of oxygenated hemoglobin in the tumor can become a quantitative marker of tumor response to the therapy. We have also continued our work on using polarization as a marker of structural changes. Polarization imaging allows one to separate contributions from surface specular reflection of the tissues and back scattered light from the deeper layers. As a result, hidden subsurface structures can be visualized to assess the transition of tissue from the healthy to diseased state. Correlation and filtering algorithms have been designed to improve image quality in the presence of noise and uncover regions of high statistical similarity, related, for example to the tissue collagen structure. To improve our previous setup, the polarization imaging system was upgraded to make possible automatic changes in focal length (stack of several co- and cross-polarized images at different focal planes). The system was tested on two healthy female volunteers over four weeks to observe the changes in the cervical structure at different time points of the menstrual cycle.